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From Reads to a Matrix

Pipeline Execution

Section titled “Pipeline Execution”

nf-core/scrnaseq

Section titled “nf-core/scrnaseq”
Terminal window
nextflow run nf-core/scrnaseq \
    -profile singularity \
    --input samplesheet.csv \
    --outdir results \
    --genome GRCh38 \
    --aligner cellranger

Cell Ranger (10x Genomics)

Section titled “Cell Ranger (10x Genomics)”
Terminal window
cellranger count \
    --id=sample1 \
    --transcriptome=/ref/refdata-gex-GRCh38 \
    --fastqs=/data/fastqs \
    --sample=sample1

Key Outputs

Section titled “Key Outputs”
sample1/
├── outs/
│   ├── filtered_feature_bc_matrix/    # Use this
│   │   ├── barcodes.tsv.gz
│   │   ├── features.tsv.gz
│   │   └── matrix.mtx.gz
│   ├── raw_feature_bc_matrix/         # Includes empty droplets
│   ├── web_summary.html               # QC report
│   └── cloupe.cloupe                  # Loupe Browser file

Count Matrix Structure

Section titled “Count Matrix Structure”

The Data

Section titled “The Data”
            Cell_1  Cell_2  Cell_3  ...
Gene_A      0       5       0
Gene_B      12      0       3
Gene_C      0       0       0
...
  • Rows: Genes (~20,000)
  • Columns: Cells (1,000-100,000)
  • Values: UMI counts (mostly zeros!)

Datasets for Practice

Section titled “Datasets for Practice”

PBMC 3k/10k

Section titled “PBMC 3k/10k”

Standard tutorial dataset from 10x Genomics:

  • Peripheral blood mononuclear cells
  • Well-characterized cell types
  • Available at: 10x datasets
# Load PBMC 3k directly
library(SeuratData)
InstallData("pbmc3k")
data("pbmc3k")

Tabula Muris

Section titled “Tabula Muris”

Mouse organ atlas:

  • Multiple tissues
  • Both 10x and Smart-seq2
  • Great for reference mapping

Human Cell Atlas

Section titled “Human Cell Atlas”

Comprehensive human reference: